The study is supported by the Ministry of Education, Singapore (AcRF Tier1-RG40/13 and Tier2-MOE2013-T2-2-038). The manuscript was edited by Amy Sullivan from Obrizus Communications.

All mice are bred and maintained under specific pathogen-free conditions and all mouse protocols are conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee.
1. Preparation of Buffers and Reagents
<ol>
	<li>Prepare complete Roswell Park Memorial Institute (RPMI) medium (10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 IU/ml)/streptomycin (100 &#181;g/ml), 55 &#181;M 2-mercaptoethanol).</li>
	<li>Prepare 20x Balanced Salt Solution (BSS) Stock 1 and Stock 2, Separately.
	<ol>
		<li>Prepare 20x BSS stock 1 (111 mM dextrose, 8.8 mM potassium phosphate, 26.7 mM sodium phosphate dibasic in 1 L sterile water) and add 40 ml 0.5% Phenol Red to 20x BSS stock 1 before final volume adjustment. Sterile filter using 0.2 &#181;m filter before storing at 4 &#176;C.</li>
		<li>Prepare 20x BSS stock 2 (25.8 mM calcium chloride dihydrate, 107 mM potassium chloride, 2.73 M sodium chloride, 19.6 mM magnesium chloride hexahydrate, 16.6 mM magnesium sulfate in 1 L sterile water). Sterile filter using 0.2 &#181;m filter before storing at 4 &#176;C.</li>
		<li>To prepare 1x BSS for experimental use, dilute 50 ml BSS stock 1 and 50 ml BSS stock 2, separately, in 400 ml sterile water each. Combine both diluted solutions, adjust to pH 7, and add 20 ml FBS (2%). Top up to 1 L using sterile water and sterile filter using 0.2 &#181;m filter. 
		NOTE: BSS stock solutions should be prepared separately because the mixing of concentrated BSS stocks directly could result in precipitation.</li>
	</ol>
	</li>
	<li>Red Blood Cell (RBC) Lysis Buffer
	<ol>
		<li>To prepare RBC lysis buffer, mix 9 parts stock ammonium chloride (155 mM ammonium chloride in sterile water) to 1 part stock Tris-base (130 mM Tris(hydroxymethyl)aminomethane in sterile water, pH 7.65) before use. 
		NOTE: Store sterile stock solutions of ammonium chloride and Tris-base at 4 &#176;C. Prepare lysis buffer fresh to ensure efficient lysis of RBCs.</li>
	</ol>
	</li>
</ol>
2. Generation of Lymphocyte Suspension from Spleen or Lymph Nodes
NOTE: It is important to prepare all reagents and equipment required for the experiment before mouse euthanasia and to generate single cell suspensions of lymphocytes as soon as possible to maintain high cell viabilities.
<ol>
	<li>Euthanize experimental mouse by cervical dislocation or CO2 asphyxiation. 
	NOTE: From this step onwards, all experimental procedures should be performed aseptically.</li>
	<li>Dip the entire mouse into 70% ethanol before making any incisions. Remove the spleen and lymph nodes aseptically8, and place them in separate 15 ml tubes containing 5 ml ice-cold RPMI/FBS (RPMI with 2% FBS) or BSS/FBS (from step 1.2.3). 
	NOTE: Since BSS prevents efficient RBC lysis, use RPMI for the preparation of the splenic cell suspension and switch to BSS after RBC lysis.</li>
	<li>To generate a single cell suspension from spleen or lymph nodes, place the organ(s) in between two pieces of sterile 100 &#181;m cell strainer mesh in a petri dish containing 2 ml ice-cold RPMI/FBS or BSS/FBS. Using the plunger of a 1 ml syringe, mash the organ(s) until it has been torn into very fine parts.</li>
	<li>Transfer the cell suspension to a 15 ml tube and wash the cell strainer mesh with ice-cold RPMI/FBS or BSS/FBS. Collect the remaining cell suspension, add it to the same 15-ml tube, and spin down at 453 x g for 5 min at 4 &#176;C. Remove the supernatant. 
	NOTE: Re-suspend the pelleted cells by flicking the tube with fingers before adding RBC lysis buffer or medium in the subsequent steps.</li>
	<li>Prepare room temperature (RT) RBC lysis buffer during centrifugation of the cell suspension (see step 1.3.). After pelleting the cells and removing the supernatant, re-suspend the cells with 1 ml RBC lysis buffer for every 108 cells. Incubate the lysis reaction at RT for 3-4 min.</li>
	<li>Stop RBC lysis with 14 ml ice-cold BSS/FBS and spin down at 453 x g for 5 min at 4 &#176;C.</li>
</ol>
3. Purification of B and T Cells
<ol>
	<li>Purification of B Cells
	<ol>
		<li>Count the cells using a hemocytometer. Re-suspend up to 108 splenic cells in 300 &#181;l BSS/FBS and add 50 &#181;l anti-CD43 magnetic microbeads9,10. To remove dead cells, add 30 &#181;l Annexin V magnetic beads. Incubate the cell suspension in a 4 &#176;C refrigerator for 30 min.</li>
	</ol>
	</li>
	<li>Purification of T Cells
	<ol>
		<li>Count the cells using a hemocytometer. Re-suspend up to 108 cells in non-T cell depletion antibody cocktail (biotinylated antibodies against CD19, B220, Gr-1, TCR-&#947;&#948;, CD49b, CD11c, CD11b, Ter119 and CD4 or CD8 depending on the target cell population to be purified), diluted 1:200 in 200 &#181;l BSS/FBS4,5. Incubate the cell suspension in a 4 &#176;C refrigerator for 15 min.</li>
		<li>After incubation, add 10 ml BSS/FBS to wash the cells and spin down at 453 x g for 5 min at 4 &#176;C. Remove the supernatant and re-suspend the cells in 165 &#181;l BSS/FBS with 30 &#181;l streptavidin microbeads and 15 &#181;l Annexin V magnetic beads. Incubate the cell suspension in a 4 &#176;C refrigerator for 30 min. 
		NOTE: To ensure even labeling with the magnetic microbeads, incubate the cells with microbeads for 15 min, then mix the cell suspension gently by tapping the 15 ml tube and incubate another 15 min during step 3.1.1. or 3.2.2.</li>
	</ol>
	</li>
	<li>Preparation of Separation Column for Cell Purification
	<ol>
		<li>Prepare an unused separation column during microbead labeling of the cells (step 3.1.1. or 3.2.2.). Pre-warm BSS (without FBS) to RT and use 2 ml to wash and equilibrate the column aseptically. After equilibration with BSS, the washed column should not be allowed to dry out. 
		NOTE: We use the LS column instead of the recommended LD column due to its re-usability (see step 3.4.).</li>
		<li>After labeling with the magnetic beads, add 14 ml BSS/FBS to wash the cells and spin down at 453 x g for 5 min at 4 &#176;C. Remove the supernatant and re-suspend the cells in 1-3 ml RT BSS.</li>
		<li>Attach a sterile 21 G needle to the tip of the column to reduce the flow rate during the process of purification. Load the cell suspension onto the equilibrated column and collect the flow through containing the purified target cells. 
		NOTE: Avoid introducing bubbles into the column while loading.</li>
		<li>Wash the column once with 1 ml BSS/FBS and collect the flow through containing the purified target cells. Reload the column with the flow through once again. Collect the flow through after the second loading in the same 15 ml tube.</li>
		<li>Wash the column 3 times with 1 ml BSS/FBS and collect the flow through containing the purified target cells. Thereafter, add 5 ml BSS/FBS to the column and, with a plunger, flush the magnetically labeled cells out of the column into a new 15 ml tube.</li>
		<li>Check the purity of the cells collected by flow cytometry using antibodies that bind to surface antigens of purified B or T cells4,5.</li>
	</ol>
	</li>
	<li>Re-using the Separation Column 
	NOTE: The LS column can be reused up to 4 times without affecting purification efficiency.
	<ol>
		<li>Wash the column 3 times with 5 ml phosphate buffered saline (PBS) and 3 times with 5 ml distilled water from the top using the plunger.</li>
		<li>Wash the column with 5 ml 70% ethanol and dry the column extensively using an air tap to prevent the buildup of rust in the column.</li>
		<li>To prepare a used LS column for a separate purification experiment, wash the column from the bottom up with 5 ml 70% ethanol using the syringe adaptor. Next, wash the column from the bottom up twice with 5 ml sterile PBS, followed by 5 ml PBS once from the top of the column. Add 2 ml RT BSS to equilibrate the column and then proceed to loading the column with labeled cells.</li>
	</ol>
	</li>
</ol>
4. CFSE Labeling and Stimulation
NOTE: Purified cells can be subjected to a variety of in vitro and in vivo functional assays. Here, we use purified T cells to determine the T cell stimulation capability of APCs5.
<ol>
	<li>Pre-warm labeling solution (0.1% FBS in PBS) to 37 &#176;C prior to CFSE loading. 
	NOTE: Using a low percentage of FBS in PBS reduces cell death during CFSE loading and minimizes cell loss during centrifugation. However, too much FBS can interfere with CFSE loading.</li>
	<li>Wash purified cells twice with labeling solution, then re-suspended at 2 x 107 cells/ml in pre-warmed labeling solution in a 15 ml tube.</li>
	<li>Prepare 10 &#181;M CFSE solution (1:500 dilution of 5 mM CFSE stock solution) in pre-warmed labeling solution. CFSE solution should be freshly prepared each time to achieve optimal labeling.</li>
	<li>To load cells with CFSE, add 1 part cell suspension to 1 part 10 &#181;M CFSE solution in a 15-ml tube and incubate in the dark for 10 min at 37 &#176;C. A final concentration of 5 &#181;M CFSE is used to label 1 x 107 cells/ml.</li>
	<li>Invert the tube every 2 min to ensure a homogenous mixture of cells during CFSE loading.</li>
	<li>To stop the reaction, add several volumes of ice-cold complete RPMI medium and spin down at 453 x g for 5 min at 4 &#176;C. Upon successful CFSE loading, the cell pellet will appear yellowish.</li>
	<li>Wash CFSE loaded cells one more time with ice-cold complete RPMI medium and spin down at 453 x g for 5 min at 4 &#176;C before using for in vitro culturing or in vivo stimulation.</li>
</ol>
5. In Vitro Stimulation
<ol>
	<li>Prepare a 2x stock solution of stimuli (2x stimuli stock solution) immediately before use so that 100 &#181;l of 2x stimuli stock solution can be added to 100 &#181;l of cells to a final volume of 200 &#181;l per well in a 96-well plate.</li>
	<li>If a plate coated with stimuli (IgM or CD40 for B cells or CD3 and CD28 for T cells) is required, dilute the stimuli in PBS and pre-coat the culture plate at 4 &#176;C overnight. Alternatively, the culture plate can be coated at 37 &#176;C for 1 hr on the day of the experiment. Wash the coated plate twice with PBS (Do not allow the plate to dry out at any time).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>For B cells&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimuli&#160;</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>F(ab&#8217;)2 goat anti-mouse IgM</td>
			<td>0.6-2.4 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Anti-mouse CD40 mAb</td>
			<td>0.5-2 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Recombinant mouse IL-4</td>
			<td>25 U/ml&#160;</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide</td>
			<td rowspan="2">0.1-10 &#181;g/ml</td>
		</tr>
		<tr>
			<td>(LPS) from E. coli Serotype 055:B5</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>For T cells&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimuli&#160;</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Anti-CD3 (plate coated)</td>
			<td>2-10 &#181;g /ml</td>
		</tr>
		<tr>
			<td>(50 &#181;l/well for coating)</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD28 (plate coated)&#160;</td>
			<td>2 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Recombinant IL-2</td>
			<td>40 U/ml</td>
		</tr>
		<tr>
			<td>PDBu (Phorbol ester)</td>
			<td>5-50 ng/ml</td>
		</tr>
		<tr>
			<td>A23187 (Calcium ionophore)</td>
			<td>250 ng/ml</td>
		</tr>
	</tbody>
</table>
Table 1: Concentrations of stimuli used to stimulate lymphocytes in in vitro culture.
<ol start="3">
	<li>For CFSE-labeled B cells, re-suspend to 3 x 106 cells/ml in complete RPMI medium and culture in triplicate with 3 x 105 cells/well in 96-well flat bottom plates for 72 hr.</li>
	<li>For CFSE-labeled T cells, re-suspend to 0.5-3 x 106 cells/ml in complete RPMI medium and culture in triplicate with 0.5-3 x 105 cells/well in 96-well round bottom plates for 48 or 72 hr.</li>
</ol>
6. In Vivo Stimulation
<ol>
	<li>For in vivo stimulation, adoptively transfer 4 x 106 CFSE-labeled T cells per mouse (intravenously (i.v.) in 200 &#181;l PBS) into each MHC-matched recipient mouse. 
	NOTE: In this protocol, CFSE-labeled T cells can be adoptively transferred using tail vein or retro-orbital injection as these cells will home to lymphoid organs such as the spleen and lymph nodes.</li>
	<li>Challenge the recipient mice one day later with the antigen. 
	NOTE: In this example, we use ovalbumin (OVA protein, 50 &#181;g/mouse) as the antigen because OVA-specific, T cell receptor (TCR)-transgenic T cells were adoptively transferred into recipient mice. Prepare OVA protein in sterile PBS and inject 100 &#181;l of OVA protein/PBS or PBS control, via subcutaneous injection (s.i.), into each recipient mouse5.</li>
	<li>Harvest and generate single cell suspensions from lymphoid organs (lymph nodes and spleens) of recipient mice 3 days after immunization with OVA protein or PBS. Separate lymph nodes into proximal lymph nodes (pLN), which includes axillary, brachial and superficial cervical lymph nodes) and distal lymph nodes (dLN), which includes mesenteric, popliteal, inguinal, lumbar, and caudal lymph nodes. Stain cells using the appropriate FACS antibodies to check for T cell proliferation. 
	NOTE: In this CFSE cell tracking experiment, CFSE-labeled T cells from PBS-injected control mice establish a baseline fluorescence for non-dividing cells. Cell divisions of proliferating, antigen-stimulated, CFSE-labeled cells are visualized by measuring fluorescence peaks12,13. With the PBS control, the number of cell divisions of proliferating, CFSE-labeled T cells can be determined12,13.</li>
	<li>Analyze the CFSE-labeled cell proliferation data by comparing the number of cell divisions or peaks between samples (Figures 1 and 2). 
	NOTE: For example, CFSE-labeled, OVA-specific T cells adoptively transferred into recipient mice receiving the OVA antigen will undergo active proliferation compared to the PBS-injected control mice5,12. Furthermore, it is noteworthy to point out that there are many ways to analyze CFSE cell proliferation data, as demonstrated by Hawkins and colleagues14.</li>
</ol>

<ol>
	<li>Nikolich-&#381;ugich, J., Slifka, M. K., Messaoudi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+important+facets+of+T-cell+repertoire+diversity.">The many important facets of T-cell repertoire diversity.</a> Nat. Rev. Immunol. 4 (2), 123-132 (2004).</li><li>LeBien, T. W., Tedder, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+lymphocytes:+how+they+develop+and+function.">B lymphocytes: how they develop and function.</a> Blood. 112 (5), 1570-1580 (2008).</li><li>Brownlie, R., Zamoyska, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+signaling+networks:+branched,+diversified+and+bound.">T cell receptor signaling networks: branched, diversified and bound.</a> Nat. Rev. Immunol. 13 (4), 257-269 (2013).</li><li>Neo, W. H., Lim, J. F., Grumont, R., Gerondakis, S., Su, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-rel+regulates+ezh2+expression+in+activated+lymphocytes+and+malignant+lymphoid+cells.">C-rel regulates ezh2 expression in activated lymphocytes and malignant lymphoid cells.</a> J. Biol. Chem. 289 (46), 31693-31707 (2014).</li><li>Gunawan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+methyltransferase+Ezh2+controls+cell+adhesion+and+migration+through+direct+methylation+of+the+extranuclear+regulatory+protein+talin.">The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin.</a> Nat Immunol. 16 (5), 505-516 (2015).</li><li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J. Immunol. Methods. 171 (1), 131-137 (1994).</li><li>Cabatingan, M. S., Schmidt, M. R., Sen, R., Woodland, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na&#239;ve+B+lymphocytes+undergo+homeostatic+proliferation+in+response+to+B+cell+deficit.">Na&#239;ve B lymphocytes undergo homeostatic proliferation in response to B cell deficit.</a> J. Immunol. 169 (12), 6795-6805 (2002).</li><li>Bedoya, S. K., Wilson, T. D., Collins, E. L., Lau, K., Larkin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Th17+differentiation+of+na&#239;ve+CD4+lymphocytes.">Isolation and Th17 differentiation of na&#239;ve CD4 lymphocytes.</a> J. Vis. Exp. (79), e50765 (2013).</li><li>Su, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ezh2+controls+B+cell+development+through+histone+h3+methylation+and+Igh+rearrangement.">Ezh2 controls B cell development through histone h3 methylation and Igh rearrangement.</a> Nat. Immunol. 4 (2), 124-131 (2003).</li><li>Mecklenbr&#228;uker, I., Saijo, K., Zheng, N., Leitges, M., Tarakhovsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C&#948;+controls+self-antigen-induced+B-cell+tolerance.">Protein kinase C&#948; controls self-antigen-induced B-cell tolerance.</a> Nature. 416 (6883), 860-865 (2002).</li><li>Rush, J. S., Hodgkin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+activated+via+CD40+and+IL-4+undergo+a+division+burst+but+require+continued+stimulation+to+maintain+division,+survival+and+differentiation.">B cells activated via CD40 and IL-4 undergo a division burst but require continued stimulation to maintain division, survival and differentiation.</a> Eur. J. Immunol. 31 (4), 1150-1159 (2001).</li><li>Quah, B. J. C., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nat. Protoc. 2 (9), 2049-2056 (2007).</li><li>Quah, B. J. C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+methods+for+measuring+lymphocyte+proliferation+in+vitro+and+in+vivo+using+CFSE-like+fluorescent+dyes.">New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.</a> J. Immunol. Methods. 379 (1-2), 1-14 (2012).</li><li>Hawkins, E. D., Hommel, M., Turner, M. L., Battye, F. L., Markham, J. F., Hodgkin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+lymphocyte+proliferation,+survival+and+differentiation+using+CFSE+time-series+data.">Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data.</a> Nat. Protoc. 2 (9), 2057-2067 (2007).</li><li>Tomlinson, M. J., Tomlinson, S., Yang, X. B., Kirkham, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation:+Terminology+and+practical+considerations.">Cell separation: Terminology and practical considerations.</a> J. Tissue Eng. 4 (1), 1-14 (2013).</li></ol>

The authors have nothing to disclose.

In this protocol, we demonstrate a procedure for purifying lymphocytes from lymphoid organs. Cell purification using magnetic bead sorting is a fast and simple method that yields viable, highly purified target cells.
Critical Steps within the Protocol
Cell viability and cell yield
Maintaining viability of hematopoietic lineage cells in vitro is critical to ensuring successful and reproducible experiments. Chemical ...

Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the na&#239;ve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells. 
Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors. 
Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify na&#239;ve B cells or T cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</td>
		</tr>
		<tr>
			<td>RPMI 1640 (without L-Glutamine)</td>
			<td>Gibco</td>
			<td>31870025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td></td>
			<td></td>
			<td>Heat inactivated&#160;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140114</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD43 magnetic microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-801</td>
			<td>Mix well prior use</td>
		</tr>
		<tr>
			<td>Streptavidin microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td>Mix well prior use</td>
		</tr>
		<tr>
			<td>Anti-Annexin V magnetic beads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-201</td>
			<td>Mix well prior use</td>
		</tr>
		<tr>
			<td>MACS LD&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well U-bottom sterile culture plate</td>
			<td>Greiner Bio-one</td>
			<td>650180</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well F-bottom sterile culture plate</td>
			<td>Greiner Bio-one</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m cell strainer mesh</td>
			<td></td>
			<td></td>
			<td>To sterilize using UV radiation prior use</td>
		</tr>
		<tr>
			<td>0.2 &#956;m&#160; sterile disposable filter units</td>
			<td>Nalgene</td>
			<td>567-0020</td>
			<td>Can be substituted with any sterile filter device</td>
		</tr>
		<tr>
			<td>CellTrace Violet</td>
			<td>Invitrogen</td>
			<td>C34557</td>
			<td>CTV for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>CellTrace Yellow</td>
			<td>Invitrogen</td>
			<td>C34567</td>
			<td>CTY for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>CellTrace Far Red</td>
			<td>Invitrogen</td>
			<td>C34564</td>
			<td>CTFR for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>Cell Proliferation Dye eFluor 670</td>
			<td>eBioscience</td>
			<td>65-0840</td>
			<td>CPD670 for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>PKH26</td>
			<td>Sigma Aldrich</td>
			<td>PKH26GL</td>
			<td>PKH26, alternative to CFSE</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Sigma Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma Aldrich</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma Aldrich</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma Aldrich</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck Millipore</td>
			<td>S7653</td>
			<td>Can use from other sources</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma Aldrich&#160;</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)</td>
			<td>Molecular Probes</td>
			<td>C-1157</td>
			<td>Reconstitute in DMSO</td>
		</tr>
		<tr>
			<td>Phorbol 12,13-dibutyrate (PBDU, Phorbol ester)</td>
			<td>Sigma Aldrich</td>
			<td>P1269</td>
			<td></td>
		</tr>
		<tr>
			<td>A23187 (Calcium ionophore)</td>
			<td>Sigma Aldrich</td>
			<td>C7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies and recombinant protein</td>
		</tr>
		<tr>
			<td>CD11b biotin (clone m1/70)</td>
			<td>Biolegend</td>
			<td>101204</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD11c biotin (clone N418)</td>
			<td>Biolegend</td>
			<td>117304</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>Gr-1 biotin (clone RB6-8C5)</td>
			<td>Biolegend</td>
			<td>108404</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>Ter119 biotin (clone Ter119)</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>TCR-&#947;&#948; biotin (clone GL-3)</td>
			<td>Biolegend</td>
			<td>118103</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD19 biotin (clone 6D5)</td>
			<td>Biolegend</td>
			<td>115504</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>B220 biotin (clone RA3-6B2)</td>
			<td>Biolegend</td>
			<td>103204</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD49b biotin (clone DX5)</td>
			<td>Biolegend</td>
			<td>108904</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD4 biotin (clone GK1.5)</td>
			<td>Biolegend</td>
			<td>100404</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD8 biotin (clone 53-6.7)</td>
			<td>Biolegend</td>
			<td>100704</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>F(ab&#8217;)2 goat anti-mouse IgM (plate coated)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>115-006-075</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD40 mAb (plate coated)</td>
			<td>Pharmingen&#160;</td>
			<td>553722</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Recombinant IL-4</td>
			<td>ProSpec&#160;</td>
			<td>Cyt-282</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from E. coli Serotype 055:B5</td>
			<td>Sigma Aldrich</td>
			<td>L-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD3 (clone clone OKT3) (plate coated)</td>
			<td>eBioscience&#160;</td>
			<td>16-0037-85</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Anti-CD28 (clone clone 37.51 ) (plate coated)&#160;</td>
			<td>eBioscience&#160;</td>
			<td>16-0281-85</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Recombinant IL-2</td>
			<td>ProSpec</td>
			<td>Cyt-370</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from chicken egg white, Ovalbumin</td>
			<td>Sigma Aldrich</td>
			<td>A7641</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and activation of murine lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Magnetic cell purification of lymphocytes allows users to purify a target cell population in a relatively short amount of time. Using our depletion protocol, we were able to increase the percentage of CD8 T cells (OT-I in recombination-activating gene-1 (RAG-1)-deficient mice) from 72.8% (before purification) to 94.2% (after purification; Figure 1A)4,5. These purified lymphocytes can then be used for downstream functional assays to determine lymphocyte prolifer...

Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

Isolation and Activation of Murine Lymphocytes

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

isolation-and-activation-of-murine-lymphocytes

Research

JoVE Journal

Immunology and Infection

20.9K Views.  Nanyang Technological University. Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Video: Isolation and Activation of Murine Lymphocytes

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...